Thyroid cancer is the most common endocrine malignancy. Most of the patients have an indolent course, but for fear of more aggressive diseases such as distant metastasis, have nonetheless been treated with over-aggressive standard treatment, which ironically is not really effective towards those truly aggressive forms of thyroid cancer. Better understanding of the genetic basis of distant metastasis is essential for more accurate risk stratification and more effective treatment, but this effort has been limited by the lack of clinical samples. Our research group has previously established a method to use Sleeping Beauty (SB) transposon- based random insertional mutagenesis for study of cancer genetics. Using this method, we have recently created a mouse model of advanced thyroid cancer with a high incidence for distant metastasis. In the research proposed here, we will use this model to answer a fundamental question regarding how metastatic phenotypes originate from the primary DTC and test the hypothesis that the metastatic traits take shape in a subpopulation of cells in the primary tumor from newly acquired mutations which offer selection advantage for distant metastasis (clonal selection model). This question will mainly be addressed by comparing the SB insertion sites in the primary tumors and the metastatic lesions. The first aim attempts to isolate the clonal origin of a metastatic lesion in the primary tumor, after establishing unique clonal SB insertion sites in the metastatic lesion as its molecular signature, and then determine if this clone has acquired any novel phenotypes that favor metastasis by studying its expression profile. The second aim will analyze the SB insertion sites at the population level in both primary and metastatic DTC to decide if there are any common insertion sites (CIS) specific for metastatic lesion (met-CIS), which would indicate newly acquired mutations and phenotypes in the metastatic lesions, or CISs enriched in primary tumors with significant metastasis (premet-CIS), which would suggest genes/pathways predisposing to metastasis when altered in the primary tumor. Also in third aim, we will try to validate the genes associated with metastasis found in the aim #2 by looking for their alterations in advanced human thyroid cancer samples, and test their oncogenic and metastatic properties in vitro and in vivo. The results of these experiments will be the basis of the future translationa research to develop molecular markers and treatment targets for metastatic thyroid cancer.